Microparticle fractionation

ABSTRACT

We describe a method of monitoring the state of a cell, tissue, organ or organism. The method comprises establishing, for a sample of micro-particles from the cell, tissue, organ or organism, a ratio. The ratio is of a selected polypeptide in microparticles which comprise GM1 gangliosides, preferably which bind to Cholera Toxin B (CTB) (“GM1 ganglioside microparticle polypeptide”) to the selected polypeptide in microparticles which comprise exposed phosphotidylserine, preferably which bind to Annexin V (“Annexin V microparticle polypeptide”). The GM1 ganglioside microparticle polypeptide to Annexin V microparticle polypeptide ratio so established may be indicative of the state of the cell, tissue, organ or organism.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is divisional application of U.S. application Ser. No.14/361,500 filed on May 29, 2014 which is a 35 U.S.C. § 371 NationalPhase Entry Application of International Application No.PCT/SG2012/000451 filed Nov. 30, 2012, which designates the U.S., andwhich claims benefit of Singapore Application No. 201108886-1, filed onNov. 30, 2011 and Singapore Application No. 201202838-7 filed on Apr.18, 2012 the contents of which are incorporated herein by reference intheir entireties.

FIELD

The present invention relates to the fields of medicine, cell biology,molecular biology and genetics. This invention relates to the field ofmedicine.

In particular, it relates to methods of monitoring the physiological orpathological state of a cell, tissue, organ or organism. The inventionalso relates to the diagnosis and treatment of diseases such a cancer.

Reference is made to U.S. Patent Application Nos. 60/713,992, Ser. Nos.12/065,549, 12/065,551, 60/878,222, Ser. No. 12/377,398, 61/066,671,61/227,865 and 61/257,121. Reference is also made to InternationalPatent Application Numbers PCT/GB2005/003206, PCT/SG2006/000233,PCT/SG2006/000232, PCT/SG2007/000257 and PCT/SG2009/000062.

The foregoing applications, and each document cited or referenced ineach of the present and foregoing applications, including during theprosecution of each of the foregoing applications ('application andarticle cited documents), and any manufacturers instructions orcatalogues for any products cited or mentioned in each of the foregoingapplications and articles and in any of the application and articlecited documents, are hereby incorporated herein by reference.Furthermore, all documents cited in this text, and all documents citedor reference in documents cited in this text, and any manufacturer'sinstructions or catalogues for any products cited or mentioned in thistext or in any document hereby incorporated into this text, are herebyincorporated herein by reference. Documents incorporated by referenceinto this text or any teachings therein may be used in the practice ofthis invention. Documents incorporated by reference into this text arenot admitted to be prior art.

BACKGROUND

It is well established that microparticles are secreted by differentcell types and could be different depending on the cell type and thepathophysiological conditions of the cell or cellular microenvironmentsuch as Alzheimer disease, TB infection, HIV infection, cancer, hypoxia,irradiation, oxidative stress, shearing stress, and exposure activatedcomplement complexes. Microparticles are membrane vesicles. To date,there are several types of microparticles that include exosomes,ectosomes, apoptotic bodies². These microparticles contain both proteinsand RNAs. Many of these microparticles have been shown to havebiological activities that enhance either biological activities orpathogenesis.

Bodily fluids such as urine, blood, tears, saliva, bronchoaveolar fluid,tumoral effusions, epididymal fluid, amniotic fluid and milk containmany membrane vesicles. As the shedding, type and biological activity ofmicroparticles is dependent on the cell type, their physiological stateand their cellular microenvironment, these microparticles arepotentially a good source of diagnostic or prognostic markers. Depletionof these microparticles could also be potentially therapeutic. However,the identity and origin of many of these microparticles in bodily fluidsare unknown and presumably, are also highly heterogenous.

Therefore, there is a need in the art for tools that can rapidly enrichfor these microparticles and that will improve the predictability androbustness of lipid microparticle-based biomarkers or therapeuticapplications.

SUMMARY

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of chemistry, molecular biology,microbiology, recombinant DNA and immunology, which are within thecapabilities of a person of ordinary skill in the art. Such techniquesare explained in the literature. See, for example, J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel,F. M. et al. (1995 and periodic supplements; Current Protocols inMolecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York,N.Y.); B. Roe, J. Crahtree, and A. Kahn, 1996, DNA Isolation andSequencing: Essential Techniques, John Wiley & Sons; J. M. Polak andJames O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;Oxford University Press; M. J. Gait (Editor), 1984, OligonucleotideSynthesis: A Practical Approach, Irl Press; D. M. J. Lilley and J. E.Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesisand Physical Analysis of DNA Methods in Enzymology, Academic Press;Using Antibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-314-2), 1855. Handbook of Drug Screening, edited byRamakrishna Scethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Maicel Dekker, ISBN 0-8247-0562-9); and Lab Ref: A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, 2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3. Each of these general texts is herein incorporated byreference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram showing size fractionation of serum. Serum fromhealthy individuals was passed through a sepharose 2B size exclusionspin column. The column was washed 3 times with PBS.

An aliquot from the input serum, flow through and washes was resolved bySDS/PAGE and the gel was stained with silver (upper panel) orelectroblotted on nitrocellulose for western blot hybridization using ananti-CD9 antibody (lower panel).

FIG. 2 is a diagram showing CTB affinity chromatography. The flowthrough and wash 1 fractions after size exclusion chromatography of theserum were pooled and tested for the presence of GM1 ganglioside by CTBaffinity chromatography.

The fractions were incubated with biotinylated CTB and thenstrepavidin-coupled to magnetic beads before being washed three timeswith PBS. An aliquot from the input serum, flow through (or unboundfraction) and washes was resolved by SDS/PAGE and the gel was stainedwith silver (upper panel) or electroblotted on nitrocellulose forwestern blot hybridization using an anti-CD9 antibody (lower panel).

FIG. 3 is a diagram showing sedimentation of CD9+ microparticles insucrose density gradient. Serum was sedimented in a sucrose densitygradient from 22.8% to 60% w/v. After ultracentrifugation, the gradientwas fractionated in equal volumes. Each fraction was weighed and thedensity calculated.

An aliquot from each fraction denoted by the density (g/ml) at the topof the upper panel was resolved by SDS/PAGE and the gel was stained withsilver (upper panel) or electroblotted on nitrocellulose for westernblot hybridization using an anti-CD9 antibody (lower panel).

FIG. 4 is a diagram showing Annexin V affinity chromatography. Serum wasincubated with biotinylated annexin V and then strepavidin-coupled tomagnetic beads before being washed three times with PBS.

An equivalent aliquot from the input serum, flow through (or unboundfraction) and washes was resolved by SDS/PAGE and electroblotted onnitrocellulose for western blot hybridization using an anti-CD9antibody. The Bound (10×) aliquot was 10 times more that that used ineach of the other lanes.

FIG. 5. Culture medium conditioned by Myc-transformed ES-derived MSCline (Myc-HuES9.E1) and plasma were loaded onto a sucrose densitygradient. The gradient was prepared by layering 14 sucrose solutions ofconcentrations from 23% to 60% (w/v). After loading the samples, thegradient was ultracentrifuged for 18 h at 200,000 g, 4° C.

The gradient was removed from the top in 13 fractions. The density ofeach fraction was determined by weighing 100 μL of each fraction. Therelative level of CD9+ annexin V-binding microvesicles and CD9+CTB-binding microvesicles in each fraction were determined.

FIG. 6A and FIG. 6B. Plasma from healthy individuals (H) and cardiacpatients (D) were incubated with biotinylated Annexin V (FIG. 6A) orCholera Toxin B (FIG. 6B). Microvesicles that bind either Annexin V orCholera Toxin B were extracted with streptavidin-conjugated magneticbeads. Proteins in these isolated microvesicles were assayed usingspecific antibody in an ELISA.

FIG. 7. For each biomarker in either microvesicle A or B, five to tenmicroliters of plasma from heart failure patients (CHF), healthyindividuals (Con) and AMI patients (AMI) were used. The relative levelsof BNP, Flt-1, TIMP-1, CD9 and ANP in either microvesicle A or B weredetermined by first isolating the microvesicle by affinitychromatography followed by an ELISA using antibody specific for theligand. Flt-1 and CD9 are membrane bound proteins while BNP, TIMP-1 andANP are luminal proteins.

The distribution of these proteins in microvesicle A or B of thedifferent groups of patients were analysed. CHF and AMI patients hadsignificantly higher BNP level in microvesicle B but not A, relative toCon individuals. Flt-1 in both microvesicle A and B was higher for AMIpatients to Con individuals.

However, Flt-1 in microvesicle A was lower for CHF patients and that inmicrovesicle B was not significantly different from Con individuals.Among the microvesicles in the three patient groups, only microvesicleA-associated TIMP-1 in CHF patients was significantly different i.e.higher. CD9 in microvesicle A but not B was significantly higher in CHFand AMI patients. ANP in microvesicle A was significantly higher for CHFand AMI patients. In microvesicle B, ANP A was significantly higher onlyfor AMI patients.

FIG. 8. Monitoring plasma CD9 of a case of food poisoning. Plasma froman individual with food poisoning was analysed as described above duringand 3 weeks after the event Two plasma samples from an healthyindividual taken three weeks apart served as control. The averagefluorescence in the CTB fraction and AnnV faction for patient before(A1) and three weeks after food poisoning (A2) were plotted againstthose of B1 and B2. B1 and B2 were the plasma samples taken three weeksapart from a healthy control.

FIG. 9. Development of an Assay. Plasma is first incubated withbiotinylated CTB and then with streptavidin-conjugated magnetic beads.The magnetic beads are then immobilized with a magnet and washed withPBS or a isotonic salt solution. Bound microvesicles are lysed with ageneric detergent-based cell lysis buffer.

The microvesicle contents are then biotinylated by activated biotin e.g.sulfo-NHS-Biotin. To assay for a specific protein, magnetic beadconjugated antibody specific for the protein of interest is then added.The antibody-bound protein is then immobilized by magnet and washedextensively. The protein is quantified using streptavidin-conjugated HRPand a HRP colorimetric or fluorimetric substrate.

FIG. 10. Diagram showing differential distribution of proteins inannexinV-versus CTB-binding microvesicles in plasma of pregnant women(third trimester) without or with clinical diagnosis of pre-eclampsia.

DETAILED DESCRIPTION

We describe a technology to rapidly isolate different lipidmicroparticles found in plasma that could be used to identify and/orstratify biomarkers in different microparticle sub-populations toenhance their diagnostic, prognostic or theranostic value.

We demonstrate that CD9, a tetraspannin membrane protein stratifies intotwo plasma microvesicle fractions that are differentially enriched ineither annexin V-binding phosphatidylserine or cholera toxin B chain(CTB)-binding GM1 ganglioside in human plasma. The affinity for annexinV and cholera toxin B chain is mutually exclusive. The association of amembrane protein with lipids suggests that these two fractions are lipidvesicles.

We also demonstrate that the relative levels of proteins or combinationsof proteins in annexin V- and CTB-binding subfractions in plasma aredependent on the health or pathological status of the individuals.

We observe that the relative levels of CD9 in annexin V- and CTB-bindingsubfractions in healthy individuals are similar. Individuals who are ill(or at risk of illness) e.g. food poisoning or cardiac patientsundergoing PCI or atherectomy have different distribution level of CD9in these subfractions. For example, an individual with food poisoninghas a higher level of CD9 in CTB-binding subfraction than that ofhealthy individuals while maintaining a similar level of annexinV-binding subfraction.

On the other hand, cardiac patients undergoing PCI or atherectomy have asimilar level of CD9 in CTB-binding subfraction found in healthyindividuals but a higher level of CD9 in annexin V-binding subfraction.

Measurement of the relative levels of proteins in annexin V- andCTB-binding subfractions in plasma may therefore be used to assess thehealth or pathological status of individuals.

Monitoring of States

We provide a method of monitoring the state of a cell, tissue, organ ororganism.

The method may comprise establishing, for a sample of microparticlesfrom the cell, tissue, organ or organism, a ratio of the amount of aselected polypeptide in a first type of microparticles to the amount ofthe selected polypeptide in a second type of microparticles.

The ratio may be a ratio of the amount of the polypeptide in the firsttype of microparticles as compared to the amount of the polypeptide inthe second type of microparticles, i.e., between the two types ofmicroparticles.

The first type of microparticles may be microparticles which compriseGM1 gangliosides (referred to as “GM1 ganglioside microparticles” forconvenience). The first type of microparticles may be capable of bindingto Cholera Toxin B (CTB), referred to as “CTB-microvesicles” forconvenience.

The second type of microparticles may be microparticles which compriseexposed phosphotidylserine. The second type of microparticles may becapable of binding to Annexin V (referred to as “Annexin Vmicroparticles” or “AV-microvesicles” for convenience).

For convenience, we refer to the presence, amount, mass or number etc ofthe polypeptide in the first type of microparticle, where these are GM1ganglioside microparticles, as “GM1 ganglioside microparticlepolypeptide”. Similarly, for convenience, we refer to the presence,amount, mass or number etc of the polypeptide in the second type ofmicroparticle, where these are Annexin V microparticles, as “Annexin Vmicroparticle polypeptide”. Again, for convenience, we refer to theratio of the above as a “GM1 ganglioside microparticle polypeptide toAnnexin V microparticle polypeptide ratio”.

The polypeptide may be selected from the group consisting of:tetraspanin proteins (e.g. CD9, CD81, CD63) Rab GTPases (e.g Rab 5a, Rab5b and Rab 5c, Rab-27a and Rab-27b, Rab 35), LAMP (e.g. Lamp1 andLamp2), caveolins (e.g., caveolin 1 and caveolin 2), transferrinreceptor (TRFC), Clathrin Light Chain A (CLTA), Clathrin Light Chain B(CLTB), Clathrin Heavy Chain 1 (CLTC), Tsg101, Alix, PAI-1, PLGF,Procalcitonin, S-100b, TGF beta 2 (TGFB2), TIMP1. The polypeptide maycomprise CD9.

The method may be such that the microparticles comprise CD9+microparticles. The method may be such that the microparticles comprisemicrovesicles, exosomes, ectosomes or apoptotic bodies.

In particular, we provide for a method of monitoring the state of acell, tissue, organ or organism, the method comprising establishing, fora sample of microparticles from the cell, tissue, organ or organism, aratio of: (a) a selected polypeptide in microparticles which compriseGM1 gangliosides, preferably which bind to Cholera Toxin B (CTB) (“GM1ganglioside microparticle polypeptide”); to (b) the selected polypeptidein microparticles which comprise exposed phosphotidylserine, preferablywhich bind to Annexin V (“Annexin V microparticle polypeptide”); inwhich the (a) to (b) ratio (“GM1 ganglioside microparticle polypeptideto Annexin V microparticle polypeptide ratio”) so established isindicative of the state of the cell, tissue, organ or organism; and inwhich the polypeptide is selected from the group consisting of:tetraspanin proteins (e.g. CD9, CD81, CD63) Rab GTPases (e.g Rab 5a, Rab5b and Rab 5c, Rab-27a and Rab-27b, Rab 35), LAMP (e.g. Lamp1 andLamp2), caveolins (e.g., caveolin 1 and caveolin 2), transferrinreceptor (TRFC), Clathrin Light Chain A (CLTA), Clathrin Light Chain B(CLTB), Clathrin Heavy Chain 1 (CLTC), Tsg101, Alix, PAI-1, PLGF,Procalcitonin, S-100b, TGF beta 2 (TGFB2), TIMP1, preferably in whichthe polypeptide comprises CD9.

We further provide a method for establishing that a cell, tissue, organor organism is in a particular state. The method may comprise comparinga GM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio of the cell, tissue, organ or organism (or a profilecomprising such a ratio) with a GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide ratio (or a profilecomprising such a ratio) of a cell, tissue, organ or organism known tobe in that particular state.

In addition to, or instead of, using a single polypeptide, combinationsof selected polypeptides may also be used. Accordingly, where we referto polypeptides (e.g., by comparison of amount to establish ratios),such reference should be taken to include reference to combinations ofpolypeptides, for example by establishing or comparing amounts ofcombinations of polypeptides.

The method may comprise selecting microparticles in the sample whichcomprise GM1 gangliosides. This may be done for example by selectingmicroparticles in the sample which bind to Cholera Toxin subunit B(CTB).

The method may comprise selecting microparticles in the sample whichcomprise exposed phosphotidylserine. This may be done for example byselecting microparticles in the sample which bind to Annexin V.

The method may further comprise a step of selecting microparticles bysize. The size selection step may comprise size exclusionchromatography. Where this is done, the size selection step may beconducted prior to the first step, e.g., step (a) above.

Where determination of an “amount” of a polypeptide is referred, itshould be understood to extend to the determination or establishment ofthe mass, number concentration etc of the polypeptide. Where referenceis made to a ratio polypeptide (or ratio of combination), being “higher”in a first state than a second state, this may be taken to mean that theratio is statistically different in the first state compared to thesecond state with a p value <0.01.

The ratio so established may be indicative of the state of the cell,tissue, organ or organism.

The method may be such that the state of the cell, tissue, organ ororganism comprises a physiological state, a differentiation state, adevelopment state or a metabolic state or a pathological state, such asa disease state, a human disease state, a food poisoning state, adiabetic state, an immune disorder state, a neurodegenerative disorderstate, an oncogenic state, a cancerous state or a tumour state.

For example, the state may be an Human Immunovirus (HIV) infected state,a tuberculosis (TB) infected state, a Bovine Spongiform Encephalitis(BSE) infected state, or a therapeutic state, for example, a patientundergoing treatment.

The method may be such that the state of the cell, tissue, organ ororganism comprises a state of being sick, a state of poor prognosis, astate of recovery from sickness, a state of good prognosis or a healthystate.

The method may be such that the sample is selected from the groupconsisting of: sweat, urine, blood, tears, saliva, bronchoaveolar fluid,tumoral effusions, epididymal fluid, amniotic fluid and milk.

Where the sample is of an organism, the organism may comprise any animalor plant. The organism may comprise a mammal, such as a human.

The method may be such that it comprises any combination of the above.

Polypeptides

The polypeptide may be any suitable polypeptide whose presence, amount,mass or number etc may be determined.

Such determination may be conducted by any suitable means as known inthe art, depending on the protein or polypeptide. Examples of suchdetermination methods include mass spectrometry, spectrophotometry, UVabsorption, etc.

Cholera Toxin B (CTB) may have a GenBank Accession Number ABG56900.1.

Annexin V may have a GenBank Accession Number AAB40047.1 or AAB60648.1.

The polypeptide may be selected from the group consisting of:tetraspanin proteins (e.g. CD9, CD81, CD63) Rab GTPases (e.g Rab 5a, Rab5b and Rab 5c, Rab-27a and Rab-27b, Rab 35), LAMP (e.g. Lamp1 andLamp2), caveolins (e.g., caveolin 1 and caveolin 2), transferrinreceptor (TRFC), Clathrin Light Chain A (CLTA), Clathrin Light Chain B(CLTB), Clathrin Heavy Chain 1 (CLTC), Tsg101, Alix, PAI-1, PLGF,Procalcitonin, S-100b, TGF beta 2 (TGFB2), TIMP1. The polypeptide maycomprise CD9.

CD9 may comprise a polypeptide with GenBank Accession NumberNP_001760.1; CD81 may comprise a polypeptide with GenBank AccessionNumber NP_004347.1; CD63 may comprise a polypeptide with GenBankAccession Number NP_001771.1; Rab5a may comprise a polypeptide withGenBank Accession Number NP_004153.2; Rab5b may comprise a polypeptidewith GenBank Accession Number NP_002859.1; Rab5c may comprise apolypeptide with GenBank Accession Number NP_004574.2; Rab-27a maycomprise a polypeptide with GenBank Accession Number NP_004571.2;Rab-27b may comprise a polypeptide with GenBank Accession NumberNP_004154.2; Rab35 may comprise a polypeptide with GenBank AccessionNumber NP_006852.1; Lamp1 may comprise a polypeptide with GenBankAccession Number NP_005552.3; Lamp2 may comprise a polypeptide withGenBank Accession Number NP_002285.1; Caveolin1 may comprise apolypeptide with GenBank Accession Number NP_001744.2; Caveolin2 maycomprise a polypeptide with GenBank Accession Number NP_001224.1;transferrin receptor (TFRC) may comprise a polypeptide with GenBankAccession Number NP_001121620.1; Clathrin Light Chain A (CLTA) maycomprise a polypeptide with GenBank Accession Number NP_001070145.1;Clathrin Light Chain B (CLTB) may comprise a polypeptide with GenBankAccession Number NP_001825.1; Clathrin Heavy Chain 1 (CLTC) may comprisea polypeptide with GenBank Accession Number NP_004850.1; Tsg101 maycomprise a polypeptide with GenBank Accession Number NP_006283.1; Alixmay comprise a polypeptide with GenBank Accession Number NP_037506.2;PAI1 may comprise a polypeptide with GenBank Accession NumberNP_000593.1; PLGF may comprise a polypeptide with GenBank AccessionNumber NP_002623.2; ProCalcitonin may comprise a polypeptide withGenBank Accession Number NP_001029124.1; S100b may comprise apolypeptide with GenBank Accession Number NP_006263.1; TGFB2 maycomprise a polypeptide with GenBank Accession Number NP_001129071.1; andTIMP1 may comprise a polypeptide with GenBank Accession NumberNP_003245.1.

Accordingly, we describe a method comprising selecting a polypeptide andestablishing a ratio of the mass, number, amount, etc of the selectedpolypeptide in GM1 ganglioside microparticles compared to the selectedpolypeptide in Annexin V microparticles, in a sample of microparticles.The sample of microparticles may be in or of or from etc a cell, tissue,organ or organism.

Combinations of Polypeptides

As noted above, instead of, or in addition to, detection of a singlepolypeptide, any combination of two or more polypeptides may be used.Thus, we describe a method in which a combination of two or morepolypeptides is selected and their mass, number, amount, etc detected ina first type of microparticle, as compared to their mass, number,amount, etc detected in a second type of microparticle, in a sample fromor of a cell, tissue, organ or organism. The ratio so established isindicative of the state of a cell, tissue, organ or organism.

For example, combinations of any two or more of PAI-1, PLGF,Procalcitonin, S-100b, TGF beta 2, TIMP1 may be employed. We describefor example use of any one or more of: (a) PAI1 and PLGF; (b) PAI1 andProcalcitonin; (c) PAI1 and S100b; (d) PAI1 and TGFbeta2; (e) PAI1 andTIMP1; (f) PLGF and Procalcitonin; (g) PLGF and S100b; (h) PLGF andTGFbeta2; (i) PLGF and TIMP1; (j) Procalcitonin and S100b; (k)Procalcitonin and TGFbeta2; (1) Procalcitonin and TIMP1; (m) S100b andTGFbeta2; (n) S100b and TIMP1 and (o) TGFbeta2 and TIMP1, in thedetection of a state comprising for example pre-eclampsia.

Polypeptide Profiles

The method may comprise establishing a profile comprising a plurality ofGM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratios for a plurality of selected polypeptide species. Eachof the profiles may be indicative of the state of the cell, tissue,organ or organism. In other words, more than one polypeptide species maybe used (i.e., ratios obtained for more than one polypeptide species).

Where reference is made to a ratio of GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide (or ratio ofcombination to combination), for example a CD9 polypeptide, being“higher” in a first state than a second state, this may be taken to meanthat the ratio is statistically different in the first state compared tothe second state with a p value <0.01. The same applies where amounts ofcombinations of polypeptides are determined.

Normalisation

The method may further comprise a step of normalisation. Such a step maycomprise determining or ensuring that the quantity or concentration ofany one or more proteins or polypeptides is the same across differentsamples.

A normalisation step, as applied to the methods and compositionsdescribed here, may make use of a polypeptide whose concentration isknown to be the same across any two samples.

Accordingly, the methods described here may comprise a normalisationstep. The normalisation step may comprise adjusting the level,concentration or amount of a particular polypeptide in one or moresamples. The normalisation step may be conducted on two or more samplesin which the level, concentration or amount of a particular polypeptide(prior to normalisation) are substantially different from each other.The normalisation step may be such that, following normalisation, thelevel, concentration or amount of a particular polypeptide in two ormore samples are substantially the same.

The normalisation step may comprise diluting or concentrating one orother of the two samples, to increase or decrease the level,concentration or amount of a particular polypeptide in one or bothsamples.

Alternatively, or in addition, the normalisation step may comprisedetermining, for a selected two or more samples, the ratio of the levelsconcentration or amount of a particular polypeptide between the samples.This may be achieved by reference to a reference polypeptide which isknown to have the same level, concentration or amount in each of a groupof samples of interest. The reference polypeptide may comprise one ormore of BNP, CD9 and TIMP-1.

It will be appreciated that, where the normalisation step comprises suchdetermination of ratios, concentration or dilution of samples may not beneeded.

Normalisation Using BNP Polypeptide

We have established that levels of BNP polypeptide in Annexin Vmicroparticles in a cell, tissue, organ or organism that is suffering(or is at risk of suffering) from Chronic Heart Failure (CHF) disease isnot statically different from levels of BNP polypeptide in a cell,tissue, organ or organism that is not suffering (or is at risk ofsuffering) from Chronic Heart Failure (CHF) disease, e.g., from healthyindividuals.

Similarly, we have established that levels of BNP polypeptide in AnnexinV microparticles in a cell, tissue, organ or organism that is suffering(or is at risk of suffering) from Acute Myocardial Ischemia (AMI)disease is not statically different from levels of BNP polypeptide in acell, tissue, organ or organism that is not suffering (or is at risk ofsuffering) from Acute Myocardial Ischemia (AMI) disease, e.g., fromhealthy individuals.

Accordingly, the level or concentration or quantity of BNP proteins orpolypeptides in in Annexin V microparticles in a cell, tissue, organ ororganism may be used to normalise any of the other markers usable in themethod described here, e.g., CD9 levels. Such normalisation may beeffected in either Annexin V microparticles or GM1 gangliosidemicroparticles or both.

Normalisation Using TIMP-1 and CD 9 Polypeptide

We have established that levels of TIMP-1 polypeptide (GenBank AccessionNumber NP_003245.1) and CD9 polypeptide (GenBank Accession NumberNP_001760.1) in GM1 ganglioside microparticles in a cell, tissue, organor organism that is suffering (or is at risk of suffering) from ChronicHeart Failure (CHF) disease is not statically different from levels ofTIMP-1 and CD9 polypeptide in a cell, tissue, organ or organism that isnot suffering (or is at risk of suffering) from Chronic Heart Failure(CHF) disease, e.g., from healthy individuals.

Similarly, we have established that levels of TIMP-1 polypeptide(GenBank Accession Number NP_003245.1) and CD9 polypeptide (GenBankAccession Number NP_001760.1) in GM1 ganglioside microparticles in acell, tissue, organ or organism that is suffering (or is at risk ofsuffering) from Acute Myocardial Ischemia (AMI) disease is notstatically different from levels of TIMP-1 and CD9 polypeptide in acell, tissue, organ or organism that is not suffering (or is at risk ofsuffering) from Acute Myocardial Ischemia (AMI) disease, e.g., fromhealthy individuals.

Accordingly, the level or concentration or quantity of TIMP-1 or CD9proteins or polypeptides (or both) in in GM1 ganglioside microparticlesin a cell, tissue, organ or organism may be used to normalise any of theother markers usable in the method described here. Such normalisationmay be effected in either Annexin V microparticles or GM1 gangliosidemicroparticles or both.

Establishment of Cardiovascular Disease State

We demonstrate that the GM1 ganglioside microparticle CD9 polypeptide toAnnexin V microparticle polypeptide CD9 ratio of a cell, tissue, organor organism that is suffering (or is at risk of suffering) fromcardiovascular disease is higher than the cognate CD9 ratio (i.e., theGM1 ganglioside microparticle CD9 polypeptide to Annexin V microparticlepolypeptide CD9 ratio) in normal or healthy cell, tissue, organ ororganisms, i.e., those not suffering from cardiovascular disease.

We therefore describe a method of establishing the cardiovasculardisease state of cell, tissue, organ or organism of interest, preferablyan organism. Accordingly, the method described above may be such thatthe states of the cell, tissue, organ or organism comprise acardiovascular disease state and a healthy state. The polypeptide maycomprise CD9 in this case. The GM1 ganglioside microparticle CD9polypeptide to Annexin V CD9 microparticle polypeptide ratio may behigher in a cardiovascular disease state compared to a healthy state.Any combination of the above may be done.

The method may comprise providing a sample of microparticles of or fromthe cell, tissue, organ or organism of interest. The amount of CD9 inGM1 ganglioside microparticles, i.e., microparticles which comprise GM1gangliosides, preferably which bind to Cholera Toxin B (CTB) isestablished. The amount of CD9 in Annexin V microparticles, i.e.,microparticles which comprise exposed phosphotidylserine, preferablywhich bind to Annexin V is established. The ratio between the amount ofCD9 in GM1 ganglioside microparticles compared to the amount of CD9 inAnnexin V microparticles is then established. This ratio is compared tothe CD9 GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide ratio of a normal or healthy cell, tissue,organ or organism (i.e., not suffering from cardiovascular disease).

Where the ratio is higher for the cell, tissue, organ or organism ofinterest than the ratio from the “normal” cell, tissue, organ ororganism of interest, then the cell, tissue, organ or organism ofinterest is determined to be suffering, or at risk of suffering from,cardiovascular disease.

Establishment of Chronic Heart Failure (CHF) State

We demonstrate that the GM1 ganglioside microparticle CD9 polypeptide toAnnexin V microparticle polypeptide CD9 ratio of a cell, tissue, organor organism that is suffering (or is at risk of suffering) from ChronicHeart Failure (CHF) disease is higher than the cognate CD9 ratio (i.e.,the GM1 ganglioside microparticle CD9 polypeptide to Annexin Vmicroparticle polypeptide CD9 ratio) in normal or healthy cell, tissue,organ or organisms, i.e., those not suffering from Chronic Heart Failure(CHF) disease.

We therefore describe a method of establishing the Chronic Heart Failure(CHF) disease state of cell, tissue, organ or organism of interest,preferably an organism. Accordingly, the method may be such that thestates of the cell, tissue, organ or organism comprise a Chronic HeartFailure (CHF) disease state and a healthy state. The polypeptide maycomprise comprise CD9. The GM1 ganglioside microparticle CD9 polypeptideto Annexin V CD9 microparticle polypeptide ratio may be higher in aChronic Heart Failure (CHF) disease state compared to a healthy state.Any combination of the above may be done.

The method may comprise providing a sample of microparticles of or fromthe cell, tissue, organ or organism of interest. The amount of CD9 inGM1 ganglioside microparticles, i.e., microparticles which comprise GM1gangliosides, preferably which bind to Cholera Toxin B (CTB) isestablished. The amount of CD9 in Annexin V microparticles, i.e.,microparticles which comprise exposed phosphotidylserine, preferablywhich bind to Annexin V is established. The ratio between the amount ofCD9 in GM1 ganglioside microparticles compared to the amount of CD9 inAnnexin V microparticles is then established. This ratio is compared tothe CD9 GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide ratio of a normal or healthy cell, tissue,organ or organism (i.e., not suffering from Chronic Heart Failure (CHF)disease).

Where the ratio is higher for the cell, tissue, organ or organism ofinterest than the ratio from the “normal” cell, tissue, organ ororganism of interest, then the cell, tissue, organ or organism ofinterest is determined to be suffering, or at risk of suffering from,Chronic Heart Failure (CHF) disease.

Establishment of Acute Myocardial Ischemia (AMI) State

We demonstrate that the GM1 ganglioside microparticle CD9 polypeptide toAnnexin V microparticle polypeptide CD9 ratio of a cell, tissue, organor organism that is suffering (or is at risk of suffering) from AcuteMyocardial Ischemia (AMI) disease is higher than the cognate CD9 ratio(i.e., the GM1 ganglioside microparticle CD9 polypeptide to Annexin Vmicroparticle polypeptide CD9 ratio) in normal or healthy cell, tissue,organ or organisms, i.e., those not suffering from Acute MyocardialIschemia (AMI) disease.

We therefore describe a method of establishing the Acute MyocardialIschemia (AMI) disease state of cell, tissue, organ or organism ofinterest, preferably an organism. The method may be such that the statesof the cell, tissue, organ or organism comprise a Acute MyocardialIschemia (AMI) disease state and a healthy state. The polypeptide maycomprise CD9. The GM1 ganglioside microparticle CD9 polypeptide toAnnexin V CD9 microparticle polypeptide ratio may be higher in a AcuteMyocardial Ischemia (AMI) disease state compared to a healthy state. Anycombination of the above may be done.

The method may comprise providing a sample of microparticles of or fromthe cell, tissue, organ or organism of interest. The amount of CD9 inGM1 ganglioside microparticles, i.e., microparticles which comprise GM1gangliosides, preferably which bind to Cholera Toxin B (CTB) isestablished. The amount of CD9 in Annexin V microparticles, i.e.,microparticles which comprise exposed phosphotidylserine, preferablywhich bind to Annexin V is established. The ratio between the amount ofCD9 in GM1 ganglioside microparticles compared to the amount of CD9 inAnnexin V microparticles is then established. This ratio is compared tothe CD9 GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide ratio of a normal or healthy cell, tissue,organ or organism (i.e., not suffering from Acute Myocardial Ischemia(AMI) disease).

Where the ratio is higher for the cell, tissue, organ or organism ofinterest than the ratio from the “normal” cell, tissue, organ ororganism of interest, then the cell, tissue, organ or organism ofinterest is determined to be suffering, or at risk of suffering from,Acute Myocardial Ischemia (AMI) disease.

Establishment of Pre-Eclampsia State

We demonstrate that ratio of the amount of polypeptides or amounts ofcombinations of polypeptides in GM1 ganglioside microparticles toAnnexin V microparticles of a cell, tissue, organ or organism that issuffering (or is at risk of suffering) from pre-eclampsia is higher thanthe cognate polypeptide/combination ratio (i.e., the ratio of the amountof polypeptides or amounts of combinations of polypeptides in GM1ganglioside microparticles to the amount of polypeptides or amounts ofcombinations of polypeptides in Annexin V microparticles) in normal orhealthy cell, tissue, organ or organisms, i.e., those not suffering frompre-eclampsia.

We therefore describe a method of establishing the pre-eclampsia stateof cell, tissue, organ or organism of interest, preferably an organism.

Accordingly, the method described above may be such that the states ofthe cell, tissue, organ or organism comprise a pre-eclampsia state and ahealthy state. The polypeptide may be selected from the group consistingof: PAI-1, PLGF, Pro-calcitonin, S100b, TGFβ and TIMP-1.

Any combination of two or more of the above polypeptides may be used.Such a combination may be selected from the group consisting of: PAI1and PLGF; PAI1 and Procalcitonin; PAI1 and S100b; PAI1 and TGFbeta2;PAI1 and TIMP1; PLGF and Procalcitonin; PLGF and S100b; PLGF andTGFbeta2; PLGF and TIMP1; Procalcitonin and S100b; Procalcitonin andTGFbeta2; Procalcitonin and TIMP1; S100b and TGFbeta2; S100b and TIMP1and TGFbeta2 and TIMP1.

The organism may comprise a pregnant organism such as a pregnant mammal,e.g., a pregnant human. The pregnant organism may be in the firsttrimester, second trimester or third trimester.

The GM1 ganglioside microparticle polypeptide/combination to Annexin VCD9 microparticle polypeptide/combination ratio may be higher in apre-eclampsia state compared to a healthy state.

The method may comprise providing a sample of microparticles of or fromthe cell, tissue, organ or organism of interest. The amount of PAI-1,PLGF, Pro-calcitonin, S100b, TGF or TIMP-1, or a combination of PAI1 andPLGF; PAI1 and Procalcitonin; PAI1 and S100b; PAI1 and TGFbeta2; PAI1and TIMP1; PLGF and Procalcitonin; PLGF and S100b; PLGF and TGFbeta2;PLGF and TIMP1; Procalcitonin and S100b; Procalcitonin and TGFbeta2;Procalcitonin and TIMP1; S100b and TGFbeta2; S100b and TIMP1 or TGFbeta2and TIMP1 in OMI ganglioside microparticles, i.e., microparticles whichcomprise GM1 gangliosides, preferably which bind to Cholera Toxin B(CTB) is established.

The amount of the selected polypeptide or combination of polypeptides inAnnexin V microparticles, i.e., microparticles which comprise exposedphosphotidylserine, preferably which bind to Annexin V is alsoestablished. The ratio between the polypeptide or combination amounts inGM1 ganglioside microparticles compared to the polypeptide orcombination amount of CD9 in Annexin V microparticles is thenestablished. This ratio is compared to the GM1 ganglioside microparticlepolypeptide or combination amount to Annexin V microparticle polypeptideor combination amount ratio of a normal or healthy cell, tissue, organor organism (i.e., not suffering from pre-eclampsia).

Where the ratio is higher for the cell, tissue, organ or organism ofinterest than the ratio from the “normal” cell, tissue, organ ororganism of interest, then the cell, tissue, organ or organism ofinterest is determined to be suffering, or at risk of suffering from,pre-eclampsia.

Monitoring of Chances of State

According to the methods and compositions described here, the GM1ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio may be used to monitor various changes of states of acell, tissue, organ or organism.

Accordingly, we describe a method for detecting a change in state of acell, tissue, organ or organism. The method may comprise detecting achange in a GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide ratio of the cell, tissue, organ or organism(or a profile comprising such a ratio). Such a change may indicate achange in state of the cell, tissue, organ or organism.

Such cell, tissue, organ or organismal changes of state may comprisechanges of physiological states. The physiological state may comprise adifferentiation state; in other words, the GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide ratio may be used todetermine whether an cell, tissue, organ or organism is differentiatedor un-differentiated. The physiological state may comprise adevelopmental state or developmental stage.

Thus, the GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide ratio may be used to determine if a cell,tissue, organ or organism is of or from an embryonic state or stage,foetal state or stage, a neonatal state or stage, an infant state orstage, a juvenile state or stage, a toddler state or stage, anadolescent state or stage, an adult state or stage etc.

The GM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio of a cell, tissue, organ or organism may be monitoredor tracked over time intervals to establish or monitor or detect changesof state.

It will be understood that, where reference is made to establishing ordetermining the state of a cell, tissue, organ or organism, this will beunderstood to encompass establishing or determining the state, per se,as well as establishing or determining whether or not (and the extentof) cell, tissue, organ or organism being at risk of transitioning orgoing into that state. In other words, establishment of state includesestablishment of risk of entering or suffering from that state.

Detecting and Treating Disease

We describe a method of detecting a disease in a cell, tissue, organ ororganism, the method comprising obtaining a sample from or of that cell,tissue, organ or organism, performing a method as set out above on thesample, and comparing the GM1 ganglioside microparticle polypeptide toAnnexin V microparticle polypeptide ratio thereby obtained with a GM1ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio of a sample known to be of or from a diseased cell,tissue, organ or organism.

We further describe a method of treatment or prevention of a disease ina cell, tissue, organ or organism, the method comprising detecting adisease in a cell, tissue, organ or organism as set out above, andadministering a treatment for that disease to the cell, tissue, organ ororganism.

The method may be such that the sample is selected from the groupconsisting of: urine, blood, tears, saliva, bronchoaveolar fluid,tumoral effusions, epididymal fluid, amniotic fluid and milk.

The method may be such that the microparticles comprise microvesicles,exosomes, ectosomes or apoptotic bodies.

Microparticles

The microparticle may in particular comprise a vesicle such as amicrovesicle. The microparticle may comprise an exosome.

The microparticle may comprise a vesicle or a flattened sphere limitedby a lipid bilayer. The microparticle may comprise a diameter of 40-100nm. The microparticle may be formed by inward budding of the endosomalmembrane. The microparticle may have a density of ˜1.13-1.19 g/ml andmay float on sucrose gradients. The microparticle may be enriched incholesterol and sphingomyelin, and lipid raft markers such as GM1, GM3,flotillin and the src protein kinase Lyn.

Methods of isolating microparticles are known in the art and aredescribed in detail in the Examples below, as well as in documents suchas International Patent Publication WO 2009/105044.

We describe in particular a technology to rapidly isolate differentlipid microparticles found in plasma that could be used to identifyand/or stratify biomarkers in different microparticle sub-populations toenhance their diagnostic, prognostic or theranostic value.

We therefore provide a method of treating a sample containingmicroparticles, the method comprising: (a) selecting microparticles inthe sample which comprise GM1 gangliosides; and/or (b) selectingmicroparticles in the sample which comprise exposed phosphotidylserine.

The method may be such that step (a) comprises selecting microparticlesin the sample which bind to Cholera Toxin subunit B (CTB); or in whichstep (b) comprises selecting microparticles in the sample which bind toAnnexin V, or both.

The method may be such that it further comprises a step of selectingmicroparticles by size, for example, by size exclusion chromatography.

The method may be such that the microparticles comprise CD9+microparticles.

Specifically, we provide a method to rapidly isolate annexin V- andCTB-binding subfractions of lipid microparticles from plasma for thedetection of microparticle-associated proteins. Rapid isolation ofannexin V- and CTB-binding subfractions lipid microparticlesubpopulations could provide a means to stratify known diseasebiomarkers further into more defined plasma subfractions and improvetheir diagnostic, prognostic or theranostic values.

We provide a method for fractionating a sample containingmicroparticles. The method may comprise selecting an AnnexinV-subfraction of microparticles or a CTB-binding sub-fraction ofmicroparticles, or both. The method may comprise performing a method asset out above.

The method may further comprise the step of detecting and/orquantitating a membrane protein or a luminal protein, or both, in afractionated sample of microparticles.

We provide a purified sample of microparticles, in which: (a)substantially all microparticles in the sample are capable of binding tocholera toxin B (CTB) but not to Annexin V; or (b) substantially allmicroparticles in the sample are capable of binding to Annexin V but notto cholera toxin B (CTB).

Monitoring of Pathological States

The cell, tissue, organ or organismal state may comprise a pathologicalstate, such as a disease state. Thus, the GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide ratio may be used tomonitor a pathological state of a cell, tissue, organ or organism, or achange of a normal state to a pathological state.

The GM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio may be used to detect whether the cell, tissue, organor organism is in a normal (undiseased) state or a diseased state. Itmay be used to monitor the progression of a cell, tissue, organ ororganism from a normal, undiseased state to a diseased state. It may beused to monitor the stage of disease of the cell, tissue, organ ororganism. The disease may comprise any of the variety of diseases acell, tissue, organ or organism suffers or may suffer.

In general, the disease state may comprise any one or more ofhypertrophic cardiomyopathy, bacterial endocarditis, agyria, amyotrophiclateral sclerosis, dizziness, tetralogy of fallot, myocarditis,alcoholism, anemia, brachial plexus, neuropathies, hemorrhoids,congenital heart defects, alopecia areata, sickle cell anemia, mitralvalve prolapse, autonomic nervous system diseases, abnormalities,alzheimer disease, angina pectoris, rectal diseases or arrhythmogenicright.

The disease state may comprise any one or more of ventricular dysplasia,acne rosacea, amblyopia, ankylosing spondylitis, atrial fibrillation,cardiac tamponade, acquired immunodeficiency syndrome, amyloidosis,anorexia, anxiety, autism, brain neoplasms, central nervous systemdiseases, color vision defects, arteriosclerosis, back pain, breastdiseases, central nervous system infections, colorectal neoplasms,arthritis, behcet's syndrome, breast neoplasms, cerebral palsy, commoncold, asthma, bipolar disorder, burns or cervix neoplasms.

The disease state may comprise any one or more of communicationdisorders, atherosclerosis, blindness, candidiasis, charcot-mariedisease, crohn disease, attention deficit disorder, brain injuries,cataract, ulcerative colitis, cumulative trauma disorders, cysticfibrosis, developmental disabilities, eating disorders, erysipelas,fibromyalgia, decubitus ulcer, diabetes, emphysema, escherichia coliinfections, folliculitis, deglutition disorders, diabetic foot orencephalitis.

The disease state may comprise any one or more of esophageal diseases,food hypersensitivity, dementia, down syndrome, japanese encephalitis,eye neoplasms, food poisoning, dengue, dyslexia, endometriosis, fabry'sdisease, gastroenteritis, depression, dystonia, epilepsy, chronicfatigue syndrome, gastroesophageal reflux, gaucher's disease,hematologic diseases, hirschsprung disease, hydrocephalus,hyperthyroidism, gingivitis, hemophilia, histiocytosis, hyperhidrosis,hypoglycemia, glaucoma, hepatitis and hiv infections. For example, thestate may be an Human Immunovirus (HIV) infected state, a tuberculosis(TB) infected state, a Bovine Spongiform Encephalitis (BSE) infectedstate, or a therapeutic state, for example, a patient undergoingtreatment.

The disease state may comprise any one or more of hyperoxaluria,hypothyroidism, glycogen storage disease, hepatolenticular degeneration,hodgkin disease, hypersensitivity, immunologic deficiency syndromes,headache, hernia, holt-oram syndrome, hypertension, impotence,congestive heart failure, herpes genitalis, huntington's disease,pulmonary hypertension, incontinence, infertility, leukemia, systemiclupus erythematosus, maduromycosis, mental retardation, inflammation,liver neoplasms, lyme disease, malaria or inborn errors of metabolism.

The disease state state may comprise any one or more of inflammatorybowel diseases, long qt syndrome, lymphangiomyomatosis, measles,migraine, influenza, low back pain, lymphedema, melanoma, mouthabnormalities, latex allergy, obstructive lung diseases, lymphoma,meningitis, mucopolysaccharidoses or leprosy.

The disease state may comprise any one or more of lung neoplasms,macular degeneration, menopause multiple sclerosis, muscular dystrophy,myofascial pain syndromes, osteoarthritis, pancreatic neoplasms, pepticulcer, myasthenia gravis, nausea, osteoporosis, panic disorder, persiangulf syndrome, myeloma, acoustic neuroma, otitis media, paraplegia,phenylketonuria, myeloproliferative disorders, nystagmus, ovarianneoplasms or parkinson disease.

The disease state may comprise any one or more of pheochromocytoma,myocardial diseases, opportunistic infections, pain, pars planitis,phobic disorders, myocardial infarction, hereditary optic atrophy,pancreatic diseases, pediculosis, plague, poison ivy dermatitis, priondiseases, reflex sympathetic dystrophy, schizophrenia, shyness,poliomyelitis, prostatic diseases, respiratory tract diseases,scleroderma, sjogren's syndrome or polymyalgia rheumatica.

The disease state may comprise any one or more of prostatic neoplasms,restless legs, scoliosis, skin diseases, postpoliomyelitis syndrome,psoriasis, retinal diseases, scurvy, skin neoplasms, precancerousconditions, rabies, retinoblastoma, sex disorders, sleep disorders,pregnancy, rare diseases, sarcoidosis, sexually transmitted diseases,spasmodic torticollis or spinal cord injuries.

The disease state may comprise any one or more of stuttering, testicularneoplasms, trichotillomania, urinary tract, infections, spinaldystaphism, substance-related disorders, thalassemia, trigeminalneuralgia, urogenital diseases, spinocerebellar degeneration, suddeninfant death, thrombosis, tuberculosis, vascular diseases, strabismus,suicide, tinnitus, tuberous sclerosis, virus diseases, post-traumaticstress disorders, syringomyelia, tourette syndrome, turner's syndrome orvision disorders.

The disease state may comprise any one or more of psychological stress,temporomandibular joint dysfunction syndrome, trachoma, urinaryincontinence, vomiting, von willebrand's disease, renal osteodystrophy,bacterial infections, digestive system, neoplasms, bone neoplasms,vulvar diseases, ectopic pregnancy, tick-borne diseases, marfansyndrome, aging, williams syndrome, angiogenesis factor, urticaria,sepsis, malabsorption syndromes or wounds and injuries.

The disease state may comprise any one or more of cerebrovascularaccident, multiple chemical sensitivity, dizziness, hydronephrosis,yellow fever, neurogenic arthropathy, hepatocellular carcinoma,pleomorphic adenoma, vater's ampulla, meckel's diverticulum, keratoconusskin, warts, sick building syndrome, urologic diseases, ischemic opticneuropathy, common bile duct calculi, otorhinolaryngologic diseases,superior vena cava syndrome, sinusitis, radius fractures, osteitisdeformans, trophoblastic neoplasms, chondrosarcoma or reading andcarotid stenosis.

The disease state may comprise any one or more of varicose veins,creutzfeldt-jakob syndrome, gallbladder diseases, replacement of joint,vitiligo, nose diseases, environmental illness, megacolon, pneumonia,vestibular diseases, cryptococcosis, herpes zoster, fallopian tubeneoplasms, infection, arrhythmia, glucose intolerance, neuroendocrinetumors, scabies.

The disease state may comprise any one or more of alcoholic hepatitis,parasitic diseases, salpingitis, cryptococcal meningitis, intracranialaneurysm, grief, calculi, pigmented nevus, rectal neoplasms, mycoses,hemangioma, colonic neoplasms, hypervitaminosis a, nephrocalcinosis,kidney neoplasms, vitamins, carcinoid tumor, celiac disease, pituitarydiseases, brain death, biliary tract diseases or prostatitis.

The disease state may comprise any one or more of iatrogenic disease,gastrointestinal hemorrhage, adenocarcinoma, toxic megacolon, amputees,seborrheic keratosis, osteomyelitis, barrett esophagus, hemorrhage,stomach neoplasms, chickenpox, cholecystitis or chondroma.

The disease state may comprise any one or more of bacterial infectionsand mycoses, parathyroid neoplasms, spermatic cord torsion, adenoma,lichen planus, anal gland neoplasms, lipoma, tinea pedis, alcoholicliver diseases, neurofibromatoses, lymphatic diseases, elder abuse,eczema, diverticulitis, carcinoma, pancreatitis, amebiasis, pregnancycomplications, pyelonephritis, infectious mononucleosis or aneurysm.

The disease state may comprise pre-eclampsia, for example in a pregnantorganism such as a pregnant human woman. The pregnant organism may be inthe first trimester of pregnancy, the second trimester of pregnancy orthe third trimester of pregnancy. A sample may be taken of a pregnantwoman in the third trimester of pregnancy, for example, and subjectedfor analysis as described in this document to establish whether she is,or is at risk of, suffering from pre-eclampsia.

In particular, the disease state may comprise a disease such asdiabetes, immune disorders, neurodegenerative disorders or cancers ortumours. Thus, the GM1 ganglioside microparticle polypeptide to AnnexinV microparticle polypeptide ratio may be used to monitor the state of acell, tissue, organ or organism which is suffering, or is prone tosuffering, any of the diseases listed above.

Monitoring of Recovery

Protein levels, including plasma CD9 levels, may be monitored in the twodifferent microparticle populations (viz, annexin V- and CTB-bindingsubfractions) in a single individual—e.g., during an episode of foodpoisoning—to monitor the injury, recovery and baseline in a patient.

The level of CD9 in one population may indicate tissue injury, while thelevel of CD9 in the other may indicate tissue repair. By measuring therelative levels of these two populations, it can be determined if apatient has more tissue injuries (i.e. sick, poor prognosis), hasinitiated tissue repair (i.e. recovering, good prognosis) or is in goodhealth.

The ratio of GM1 ganglioside microparticle polypeptide to Annexin Vmicroparticle polypeptide may also be used to indicate prognosis of thepatient.

Biological Sample

The methods and compositions described here involve the ratio of GM1ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide secreted by a cell in order to monitor its state.Conveniently, the ratio may be determined by taking a biological samplecomprising secretions of the cell.

Where the cell is comprised in an organism, the sample may comprise anynumber of things, including, but not limited to, bodily fluids(including, but not limited to, blood, nasopbaryngeal secretions, urine,serum, lymph, saliva, anal and vaginal secretions, perspiration andsemen, of virtually any organism.

Preparation of Microparticles

CTB or Annexin V binding microparticles may be prepared from plasma (orother fluids) using the following protocol:

10 μL plasma were incubated with 0.1 μg biotinylated Cholera Toxinsubunit B (CTB) (SBL Vaccin AB) or 0.1 μg biotinylated Annexin V (AV)(BioVision) in 100 μL PBS pH 7.4 for 1 hour at 37° C. with shaking at800 rpm. In the meantime, 100 μL of Dynabeads® M-280 Streptavidin(Invitrogen) was washed three times with 100 μL PBS. After the lastwash, the plasma-CTB or plasma-AV reaction mix was added to the washedbeads and incubated with shaking at 800 rpm for 30 minutes. The beadswere immobilised with a magnet and the supernatant was removed. Thebeads were then washed thrice with 200 μl PBS and the washes wereremoved each time after immobilizing the beads with a magnet.

GM1 Ganglioside Microparticle Polypeptide to Annexin V MicroparticlePolypeptide Ratio

The GM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio may be simply calculated as a ratio of the quantity ofpolypeptide in GM1 ganglioside microparticles to the quantity ofpolypeptide in Annexin V microparticles.

Alternatively, or in addition, the quantity of another polypeptide inGM1 ganglioside microparticles to, the quantity of that polypeptide inAnnexin V microparticles known not to be changed as a result of thechange in cellular state may be used as an internal control.

Thus, instead of, or in addition to, monitoring the change in the GM1ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide ratio, the ratio of a first ratio against a second ratio maybe used for monitoring purposes. Here, the first ratio is the ratio ofGM1 ganglioside microparticle polypeptide to Annexin V microparticlepolypeptide known to be changed as a result of the change in cellularstate and the second ratio being the ratio GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide ratio known not to bechanged as a result of the change in cellular state.

GM1 Ganglioside Microparticle Polypeptide to Annexin V MicroparticlePolypeptide Ratio Profile

Alternatively or in addition to determining the ratio of GM1 gangliosidemicroparticle polypeptide to Annexin V microparticle polypeptide, aprofile comprising a plurality of GM1 ganglioside microparticlepolypeptide to Annexin V microparticle polypeptide ratios for aplurality of selected polypeptide species, each indicative of the stateof the cell, may also be established. Changes to such a profile may bemonitored as a means to monitor the state of a cell. The profile may beestablished by any means known in the art, such as by hybridisation toan array comprising a plurality of binding agents capable of binding toand distinguishing between each of the plurality of the selectedpolypeptide species.

EXAMPLES Example 1. Enrichment of CD9+ Microparticles from Serum ofNormal Individuals

In this study, we first determined if CTB will bind CD9+ microparticlesin serum. As there were no CTB-bound CD9, we determined thesedimentation density of serum CD9+ microparticles in a sucrose densitygradient and found the CD9 in the serum to have a higher sedimentationdensity than that for exosomes.

The lack of CTB binding and the high sedimentation density suggestedthat the CD9+ microparticles were apoptotic bodies. This was thenconfirmed by the binding of serum CD9 to annexin V.

Example 2. Materials and Methods—Size Exclusion Chromatography

2 mL Sepharose® 2B resins (Sigma Aldrich, Cat no. 2B300) was pipettedinto a spin column (Bio-Rad, Cat no. 732-6008) and spun d at 800 g for 1minute. The supernatant was removed and washed by adding 1 mL PhosphateBuffer Saline (PBS) followed by a 1-minute spin at 800 g. 1 mL of humanserum was then loaded and the column was spun at 800 g for 1 minute.

The flow through was collected and this process was repeated thriceusing 1 mL of PBS each time. 3 wash fractions were collected. 20 μL ofeach fraction was resolved on a 4-12% SDS/polyacrylamide gels. The gelswere either stained with SilverQuest™ Silver Staining Kit (Invitrogen,Carlsbad, Calif.) or electroblotted onto a nitrocellulose membrane. Themembrane was probed with either a 1:50 dilution of mouse anti-human CD9antibody followed by a 1:1250 dilution of a HRP-conjugated donkeyanti-mouse IgG antibody.

All antibodies were purchased from Santa Cruz. The bound antibodies werevisualized using HRP-enhanced chemiluminescent substrate (Thermo FisherScientific Inc., Waltham, Mass.) and exposure to an X-ray film.

Example 3. Materials and Methods—Cholera Toxin B Affinity Chromatography

200 μL of serum flow through from the size exclusion were incubated with0.1 g biotinylated Cholera Toxin subunit B (CTB) (Invitrogen) in 100 μLPBS pH 7.4 for 1 hour at room temperature with shaking at 800 rpm. Afterwashing 100 d of Dynabeads® M-280 Streptavidin (Invitrogen) three timeswith 100 al PBS, the reaction mix was added and incubated with shakingat 800 rpm for 30 mins.

The beads were immobilised with a magnet and the supernatant or unboundfraction was removed. The beads were then washed twice with 100 μl PBSand the washes were removed each time after immobilizing the beads witha magnet. The beads were boiled in 100 μl of a denaturing/reducingSDS-PAGE loading buffer to elute the remaining bound proteins. Equalvolume of the unbound fraction, washes, eluted and bound fraction wasresolved on 4-12% SDS-polyacrylamide gels.

The gels were either stained with silver or electroblotted onto anitrocellulose membrane. The membrane was probed with 1:50 dilution ofmouse anti-human CD9 antibody. The secondary antibody was 1:1250 of HRPconjugated donkey anti-mouse Ig G antibody. All antibodies werepurchased from Santa Cruz. The bound antibodies were visualized usingHRP-enhanced chemiluminescent substrate (Thermo Fisher Scientific Inc.,Waltham, Mass.) and exposure to an X-ray film.

Example 4. Materials and Methods—Sucrose Gradient

14 sucrose solutions with concentrations from 22.8% to 60% were preparedand layered sequentially in an ultracentrifuge tube (Beckman CoulterInc., CA) starting with the most concentrated solution. 500 μL of thehuman serum was loaded on top before ultracentrifugation for 16.5 h at200 000 g, 4° C. in a SW60Ti rotor (Beckman Coulter Inc.). Aftercentrifugation, 320 μL of 13 fractions were collected starting from thetop of the gradient.

The densities of each fractions were determined by weighing a fixedvolume. 20 μL of each fractions was resolved on 4-12% SDS-polyacrylamidegels. The gels were either stained with SilverQuest™ Silver Staining Kit(Invitrogen, Carlsbad, Calif.) or electroblotted onto a nitrocellulosemembrane. The membrane was probed with either a 1:50 dilution of mouseanti-human CD9 antibody followed by a 1:1250 dilution of aHRP-conjugated donkey anti-mouse IgG antibody. All antibodies werepurchased from Santa Cruz.

The bound antibodies were visualized using HRP-enhanced chemiluminescentsubstrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposureto an X-ray film. Fractions 9 to 13 from the sucrose gradient werepooled and dialysed against PBS pH7.4 overnight. The pooled fractionswere concentrated to 20 μL

Example 5. Materials and Methods—Annexin V Affinity Chromatography

20 μL of the pooled fractions from the sucrose gradient was mixed with20 μL of 5× Annexin V binding buffer (Calbiochem) and made up to a finalvolume of 100 μL with water. 90 μL of the pooled fraction sample wasincubated with 10 μL of Annexin V for 1 hour at room temperature withshaking at 800 rpm.

After washing 100 μl of Dynabeads® M-280 Streptavidin (Invitrogen) threetimes with 100 μl PBS, the reaction mix was added and incubated withshaking at 800 rpm for 30 mins. The beads were immobilised with a magnetand the supernatant or unbound fraction was removed. The beads were thenwashed four times with 100 μl PBS and the washes were removed each timeafter immobilizing the beads with a magnet.

The beads were boiled in 100 μl of a denaturing/reducing SDS-PAGEloading buffer to elute the remaining bound proteins. Equal volume ofthe unbound fraction, washes, eluted and bound fraction was resolved on4-12% SDS-polyacrylamide gels. The gels were electroblotted onto anitrocellulose membrane.

The membrane was probed with 1:50 dilution of mouse anti-human CD9antibody. The secondary antibody was 1:1250 of HRP conjugated donkeyanti-mouse IgG antibody. All antibodies were purchased from Santa Cruz.

The bound antibodies were visualized using HRP-enhanced chemiluminescentsubstrate (Thermo Fisher Scientific Inc., Waltham, Mass.) and exposureto an X-ray film.

Example 6. CD9 in Serum of Healthy Individuals Binds to Annexin V butnot CTB

To determine if CD9+ microparticles are present in the serum of healthyindividuals CTB, the serum was first size fractionated to enrich forlarge particles and remove abundant small serum proteins such asalbumin. The fractionated serum was then incubated CTB to determine ifCD9 in the serum binds to with annexin V or annexin V.

Example 7. Results—Large CD9+ Microparticles are Present in Serum ofHealthy Individuals by CTB

The results are shown in FIG. 1.

Size fractionation of the serum resulted in the enrichment of CD9 in theflow through and wash 1 fraction suggesting that CD9, a small proteinmust be associated with a large complex.

Example 8. Results—CD9+ Microparticles do not Bind CTB

The results are shown at the top panel of FIG. 2.

Most of the proteins did not bind to the CTB including CD 9 (lowerpanel). Therefore, CD9+ microparticles in serum of healthy individualsdid not bind CTB and did not have GM1 gangliosides. CD9+ microparticlesin serum of healthy individuals are not exosomes.

Example 9. Results—CD9+ Microparticles have a High Sedimentation Density

The results are shown in FIG. 3.

To identify the type of CD9+ microparticles in serum of healthyindividuals, the serum was fractionated on a sucrose density gradientand fractions were analysed by SDS/PAGE and western blot hybridization.CD9+ microparticles sedimented at a density of 1.181-1.226 g/ml. Thisdensity is the expected density of apoptotic bodies and not of exosomeswhich is 1.13-1.19 g/ml and (Thery, Ostrowski et al. 2009).

Example 10. Results—CD9+ Microparticles in Serum of Healthy IndividualsBind Annexin V

The results are shown in FIG. 4.

To confirm that some of CD9+ microparticles were apoptotic bodies, wedetermine if these microparticles could bind annexin V. Some of thedense CD9+ microparticles but not all could bind annexin V as CD9 wasdetected only when 10 more of the bound fraction was loaded onto thegel.

Example 11. Conclusions

Serum of healthy individuals contains CD9+ microparticles. However thesemicroparticles are not exosomes or a very small proportion of thesemicroparticles was exosomes. They do not bind CTB and therefore havelittle or no GM1 gangliosides or lipids highly enriched in lipid rafts,a hallmark feature of exosome membrane.

Furthermore, most, if not all of these microparticles are densemicroparticles with sedimentation density in sucrose of 1.181-1.226 g/mland not the 1.13-1.19 g/ml density of exosomes. Instead a significantproportion was dense apoptotic bodies that contain exposed PS. Theremaining microparticles could still be apoptotic bodies but this needsto be verified. The low level of CTB-binding CD9+ microparticles inserum of healthy individuals will increase the predictability of anybiomarkers of disease or disease susceptibility that are derived fromCTB-binding CD9+ microparticles in serum.

Given that exosomes are secreted as a vehicle of intercellularcommunication in neurons and immune cells (Smalheiser 2007; Thery,Ostrowski et al. 2009) and many disease-associated proteins have beenreported to be secreted in exosomes e.g. beta-amyloid peptides inAlzheimer's disease (Rajendran, Honsho et al. 2006), mycobacterialproteins in M. tuberculosis-infected J774 cells (Giri, Kruh et al.2010), tumor antigens (Taylor and Gercel-Taylor 2005), our observationof a low baseline of CTB-binding CD9+ microparticles in serum of healthyindividuals may an indicator of relative good health and a rise may be asentinel of disease, disease injury or increased cellular communicationfor tissue repair.

Analysis of this population of microparticles is therefore likely toyield more predictive biomarker to diagnose or prognose diseases.

Example 12. Differences Between Lipid Microvesicles in Plasma andMSC-Conditioned Medium

Materials and Methods

Culture medium conditioned by Myc-transformed ES-derived MSC line(Myc-HuES9.E1) and plasma were loaded onto a sucrose density gradient.The gradient was prepared by layering 14 sucrose solutions ofconcentrations from 23% to 60% (w/v).

After loading the samples, the gradient was ultracentrifuged for 18 h at200,000 g, 4° C. The gradient was removed from the top in 13 fractions.The density of each fraction was determined by weighing 100 μL of eachfraction. The relative level of CD9+ annexin V-binding microvesicles andCD9+ CTB-binding microvesicles in each fraction were determined.

Results

Please refer to FIG. 5.

CD9+ microvesicles in plasma are predominantly annexin V-binding whilethose in MSC-conditioned medium are predominantly cholera toxin B-chain(CTB) binding.

CTB-binding microvesicles in plasma float at two distinctive sucrosedensities 1.21-1.59 and >1.173 g/ml while CTB-binding microvesiclesfloat at one density i.e. 1.065-1.191 g/ml

Note: CD81+ microvesicles in the plasma and MSC-conditioned mediumexhibit the same distribution profile as CD9+ microvesicles.

Example 13. Distribution of Proteins in Annexin V- Versus CTB-BladingMicrovesicles in Plasma of Cardiac Patients and Healthy Individuals

Materials and Methods

Please refer to FIG. 6A and FIG. 6B.

Plasma from healthy individuals (H) and cardiac patients (D) wereincubated with biotinylated Annexin V (FIG. 6A) or Cholera Toxin B (FIG.6B). Microvesicles that bind either Annexin V or Cholera Toxin B wereextracted with streptavidin-conjugated magnetic beads. Proteins in theseisolated microvesicles were assayed using specific antibody in an ELISA.

Results

Some proteins in annexin V- and CTB-binding plasma microvesicles aredifferentially distributed between healthy individuals and patients at acardiac clinic e.g.

CD9, CD81 and TIMP1 in annexin V-binding but not CTB-binding plasmamicrovesicles are significantly higher in patients than in healthyindividuals;

ANP in CTB-binding but not annexin V-binding plasma microvesicles issignificantly higher in patients than in healthy individuals

The other proteins may exhibit a significantly different distributionprofile in the two types of microvesicles of patients vs healthyindividuals if population size is bigger, and/or the disease is morenarrowly defined.

Other biomarkers that could be used to assess differences in plasmamicrovesicles of cardiac patients versus healthy individuals are BNP,endothelin, rennin, angiotensin II, troponin, myosin, IL6, IL1, IL10,TNFα, TGFβ.

Example 14. Distribution of Proteins in Annexin V- Versus CTB-BindingMicrovesicles in Plasma of Patients with Chronic Heart Failure (CHF) andPatients with Acute Myocardial Ischemia (AMI)

To evaluate the feasibility and value of being able to fractionateplasma microvesicle for biomarker assay, we applied this technology toplasma samples from more defined patient groups: Patients with chronicheart failure (CHF) and patients with acute myocardial ischemia (AMI).The controls are plasma from healthy individuals (con) (FIG. 7).

Materials and Methods

For each biomarker in either microvesicle A or B, five to tenmicroliters of plasma from heart failure patients (CHF), healthyindividuals (Con) and AMI patients (AMI) were used. The relative levelsof BNP, Flt-1, TIMP-1, CD9 and ANP in either microvesicle A or B weredetermined by first isolating the microvesicle by affinitychromatography followed by an ELISA using antibody specific for theligand. Flt-1 and CD9 are membrane bound proteins while BNP, TIMP-1 andANP are luminal proteins.

The distribution of these proteins in microvesicle A or B of thedifferent groups of patients were analysed. CHF and AMI patients hadsignificantly higher BNP level in microvesicle B but not A, relative toCon individuals. Flt-1 in both microvesicle A and B was higher for AMIpatients to Con individuals.

However, Flt-1 in microvesicle A was lower for CHF patients and that inmicrovesicle B was not significantly different from Con individuals.Among the microvesicles in the three patient groups, only microvesicleA-associated TIMP-1 in CHF patients was significantly different i.e.higher.

CD9 in microvesicle A but not B was significantly higher in CHF and AMIpatients. ANP in microvesicle A was significantly higher for CHF and AMIpatients. In microvesicle B, ANP A was significantly higher only for AMIpatients.

Results

Please refer to FIG. 7.

Microvesicles isolated by affinity to either CTB or annexin (FIG. 7) aredifferent from each other as demonstrated by their different proteinprofile (FIG. 7). The differences are also disease specific. Forexample, BNP level in the annexin V-bound microvesicle fraction of theCHF, AMI and con was not significantly different in all three patientgroups.

However, BNP level in the CTB-bound microvesicle fraction of the CHF andAMI was significantly higher than control. On the other hand, ANP in theAnnexin (FIG. 7) fraction was significantly higher in both CHF and AMIgroups compared to the con group but ANP in the CTB fraction wassignificantly higher only in the AMI group.

Example 15. Monitoring Plasma CD9 of a Case of Food Poisoning

Plasma from an individual with food poisoning was analysed as describedabove during and 3 weeks after the event. Two plasma samples from anhealthy individual taken three weeks apart served as control.

The results are shown in FIG. 8. The average fluorescence in the CTBfraction and AnnV faction for patient before (A1) and three weeks afterfood poisoning (A2) were plotted against those of B1 and B2. B1 and B2were the plasma samples taken three weeks apart from a healthy control.

Example 16. Development of an Assay to Couple the Isolation of EitherCTB or Annexin V Binding Microvesicles to the Quantification of Membraneand Luminal Proteins in the Microvesicles

Materials and Methods

Please refer to FIG. 9.

Plasma is first incubated with biotinylated CTB and then withstreptavidin-conjugated magnetic beads. The magnetic beads are thenimmobilized with a magnet and washed with PBS or a isotonic saltsolution. Bound microvesicles are lysed with a generic detergent-basedcell lysis buffer.

The microvesicle contents are then biotinylated by activated biotin e.g.sulfo-NHS-Biotin. To assay for a specific protein, magnetic beadconjugated antibody specific for the protein of interest is then added.The antibody-bound protein is then immobilized by magnet and washedextensively.

The protein is quantified using streptavidin-conjugated HRP and a HRPcolorimetric or fluorimetric substrate.

Example 17. Conclusion

The technology could quantitatively assay for both membrane and luminalproteins in the isolated microvesicles—we could detect both membrane andluminal proteins (FIG. 7). The method for detecting both membrane andluminal proteins are schematically depicted in FIG. 8.

Example 18. Differential Distribution of Proteins in AnnexinV- VersusCTB-Binding Microvesicles in Plasma of Pregnant Women (Third Trimester)without or with Clinical Diagnosis of Pre-Eclampsia—Single Polypeptides

For each biomarker in either AnnexinV (AV)- or cholera toxin B chain(CTB)-binding microvesicles (see Tables E1 and E2 below), five to tenmicroliters of plasma from third trimester pregnant women withoutpre-eclampsia (control) and with clinically diagnosed pre-eclampsia(Pre-eclampsia).

The relative levels of PAI-1, PLGF, Pro-calcitonin, S100b, TGFβ andTIMP-1 in either AnnexinV (AV)- or cholera toxin B chain (CTB)-bindingmicrovesicles were measured as described below.

Briefly, AV- or CTB-binding microvesicles were first isolated byaffinity chromatography, the isolated microvesicles were lysed torelease proteins associated with the microvesicle and the releasedproteins were then biotinylated. The targeted protein of interest wasthen assayed using a specific antibody conjugated to HRP and afluorescent HRP substrate.

FIG. 10 demonstrates that PAI-1, PLGF, Pro-calcitonin, S100b, TGFβ andTIMP-1 in annexinV- and/or CTB-binding microvesicles in plasma ofpregnant women (third trimester) exhibited a significant quantitativedifference (p<0.05) in women without or with clinical diagnosis ofpre-eclampsia.

Therefore, these microvesicle-encapsulated proteins could be used asbiomarkers that will be diagnostic of pre-eclampsia.

Example 19. Differential Distribution of Proteins in AnnexinV- VersusCTB-Binding Microvesicles in Plasma of Pregnant Women (Third Trimester)without or with Clinical Diagnosis of Pre-Eclampsia—Combinations ofPolypeptides

The diagnostic power of these markers is greatly enhanced when they areused in combination of two proteins either in the same plasmamicrovesicle or different microvesicle (FIG. 10).

Using the logistic regression model (Moore et al, 2008), receiveroperator characteristic (ROC) curves were constructed and the area underthe curve for each marker in the CTB- or AV-bound microvesicles or acombination of two different markers were calculated (Table E1 and TableE2 below) For example, the area under the curve for PAI-1 and PLGF inthe CTB-microvesicle were 0.972 and 0.833, respectively.

When PAI-1 and PLGF were combined, the area under the curve increased to1.0 (Table E1 below).

TABLE E1 The receiver operator characteristic (ROC) curves wereconstructed for each of the biomarkers in the plasma CTB-microvesiclesand the area under the curve for each marker or a combination of twodifferent markers were calculated. Area under Curve PAI-1 .972 PLGF .833Procalcitonin 1.000 S-100b .889 TGF beta 2 .972 TIMP1 .833 PAI1 - PLGF1.000 PAI1 - Procalcitonin 1.000 PAI1 - S100b .972 PAI1 - TGFbeta2 1.000PAI1 - TIMP1 1.000 PLGF - Procalcitonin 1.000 PLGF - S100b .889 PLGF -TGFbeta2 1.000 PLGF - TIMP1 .917 Procalcitonin - S100b 1.000Procalcitonin - TGFbeta2 1.000 Procalcitonin - TIMP1 1.000 S100b -TGFbeta2 1.000 S100b - TIMP1 .917 TGFbeta2 - TIMP1 .972

Similarly, combining two or more markers in the AV-microvesicle e.g.PLGF and Procalcitonin also enhanced their respective area under curvefrom 0.889 and 0.861 respectively to 1.0 (Table E2 below).

TABLE E2 The receiver operator characteristic (ROC) curves wereconstructed for each of the biomarkers in the plasma AV-microvesiclesand the area under the curve for each marker or a combination of twodifferent markers were calculated. Area under Curve PAI-1 1.000 PLGF.889 Procalcitonin .861 S-100b .972 TGF beta 2 .972 TIMP1 1.000 PAI1 -PLGF 1.000 PAI1 - Procalcitonin 1.000 PAI1 - S100b 1.000 PAI1 - TGFbeta21.000 PAI1 - TIMP1 1.000 PLGF - Procalcitonin 1.000 PLGF - S100b 1.000PLGF - TGFbeta2 1.000 PLGF - TIMP1 1.000 Procalcitonin- S100b 1.000Procalcitonin - TGFbeta2 1.000 Procalcitonin - TIMP1 1.000 S100b -TGFbeta2 1.000 S100b - TIMP1 1.000 TGFbeta2 - TIMP1 1.000

These examples illustrate the robustness of these biomarkers to diagnosepre-eclampsia when used in combination of two or more.

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Each of the applications and patents mentioned in this document, andeach document cited or referenced in each of the above applications andpatents, including during the prosecution of each of the applicationsand patents (“application cited documents”) and any manufacturer'sinstructions or catalogues for any products cited or mentioned in eachof the applications and patents and in any of the application citeddocuments, are hereby incorporated herein by reference. Furthermore, alldocuments cited in this text, and all documents cited or referenced indocuments cited in this text, and any manufacturer's instructions orcatalogues for any products cited or mentioned in this text, are herebyincorporated herein by reference.

Various modifications and variations of the described methods and systemof the invention will be apparent to those skilled in the art withoutdeparting from the scope and spirit of the invention. Although theinvention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the claims.

The invention claimed is:
 1. A method for fractionating a samplecontaining microparticles, the method comprising: selecting an AnnexinV-binding sub-fraction of microparticles and a Cholera Toxin B(CTB)-binding sub-fraction of microparticles.
 2. The method according toclaim 1, further comprising a step of detecting or quantitating amembrane protein or a luminal protein, or both, in a fractionated sampleof microparticles.
 3. The method according to claim 2, in which themembrane protein or a luminal protein is selected from the groupconsisting of: a tetraspanin protein, CD9, CD81, CD63, a Rab GTPase, Rab5a, Rab 5b, Rab 5c, Rab-27a, Rab-27b, Rab 35, a LAMP protein, Lamp1,Lamp2, a caveolin, caveolin 1, caveolin 2, transferrin receptor (TRFC),Clathrin Light Chain A (CLTA), Clathrin Light Chain B (CLTB), ClathrinHeavy Chain 1 (CLTC), Tsg101, Alix, PAI-1, PLGF, Procalcitonin, S-100b,TGF beta 2 (TGFB2), and TIMP1.
 4. The method according to claim 1, inwhich the step of selecting an Annexin V-binding sub-fraction ofmicroparticles comprises selecting microparticles which comprise exposedphosphotidylserine.
 5. The method according to claim 1, in which thestep of selecting a CTB-binding sub-fraction of microparticles comprisesselecting microparticles which comprise GM1 gangliosides.
 6. The methodaccording to claim 1, further comprising a step of selectingmicroparticles by size.
 7. The method according to claim 6, in which thestep of selecting microparticles by size comprises size exclusionchromatography.
 8. The method according to claim 1, in which themicroparticles comprise CD9+ microparticles.
 9. The method according toclaim 1, in which the sample is selected from the group consisting of:sweat, urine, blood, tears, saliva, bronchoaveolar fluid, tumoraleffusions, epididymal fluid, amniotic fluid and milk.
 10. The methodaccording to claim 1, in which the microparticles comprisemicrovesicles, exosomes, ectosomes or apoptotic bodies.